Assays which make use of electrodes for the detection and quantitation of certain materials in a solution, e.g. immunoassays for detection and quantitation of antigens, are known in the art. For instance, electrochemiluminescence is the basis for highly sensitive detection and quantitation processes in which reactive species are electrochemically generated from stable precursors at the surface of an electrode. The electrochemically generated reactive species undergo a chemiluminescent reaction. The luminescence from the chemiluminescent reaction is used to detect or quantify ECL-active species, which have been bound to materials desired to be detected, i.e. the analyte. For example, the ECL-active species may be bound to an antibody for detection of an antigen specific to the antibody. Highly sensitive electrochemiluminescence detection systems and methods have been developed which are capable of measuring trace amounts of materials. The detection of luminescence from ECL-active labels has been used to develop assays in materials such as biochemical and biological substances and provide sensitive immunoassays and DNA probe assays.
Other types of assays making use of an electrode include, for example, amperometric and potentiometric assays.
These electrochemiluminescent detection systems have many advantages over other detection systems in that measurement is simple and rapid, no radioisotopes are used, the detection limits for ECL-active species are extremely low (200 fmol/L); the dynamic range of the ECL-active specific quantification extends over six orders of magnitude; and the ECL-active labels are extremely stable and small (.about.1000 Da) so that haptens or large molecules can be labeled and multiple labels can be coupled to proteins or oligonucleotides without affecting their immuno-reactivity, solubility or their ability to hybridize.
In addition, since the chemiluminescence requires an applied voltage, the initiation and duration of the response can be controlled by controlling the voltage applied to an electrode.
ECL detection apparatus and methods are described in more detail in the following PCT published applications: WO 86/02734, WO 87/00987, WO 88/03947. See also, Massey, Biomedical Products: Tools & Techniques, October 1992; and, Blackburn et al., Clinical Chemistry, Vol. 37, No. 9; p. 1534-1539 (1991).
PCT Published Application WO 90/05301 describes methods for performing assays based on luminescent phenomena in a homogeneous format wherein modulation of the intensity of the luminescent signal generated by the ECL-active moiety provides a means for monitoring the specific binding of an assay system. In these methods, microparticles are bound to ECL-active species of the assay components to modulate the intensity of the luminescent signal.
PCT Published Application WO 92/00982 describes methods for performing assays wherein the microparticles bound to the ECL-active species are magnetically responsive and are drawn to the electrode where the ECL-active moiety undergoes excitation by a plurality of north-south oriented magnets. This significantly enhances the ECL signal from the sample composition.
A variety of formats have been developed for assays based on the detection and quantitation of electrochemiluminescence. For example, heterogenous (one or more separations) and homogeneous (non-separation) formats have been developed for competitive assays where, for example, haptens labeled with an ECL-active moiety compete for antibody with an analyte of interest. In a heterogenous format, the free and bound fractions of the labeled component such as hapten are separated before analysis in the ECL detection apparatus.
In homogeneous formats, there is no separation of fractions before the analysis. Such a format can be used when the efficiency of ECL excitation differs considerably for the free and bound fractions of the labeled components. For example, a homogeneous immunoassay may involve adding a serum sample into a composition of specific reagents, incubating the mixture and measuring for the analyte with an immunoanalyzer without any separation or washing steps. Homogeneous immunoassays, also referred to as nonseparation immunoassays, provide the advantage of eliminating the separation steps and, thus, simplifying and decreasing the time required for the assay.
However, problems in homogeneous immunoassays can arise due to interference with the desired analyte signal by serum matrix components and unbound conjugates. Interference with the analyte signal could also arise in heterogeneous immunoassays wherein the separation(s) conducted do not remove all or significant amounts of the interfering serum matrix components and unbound conjugate components.
While the apparatus and methods taught in the prior art such as WO 90/05301 and WO 92/0982 and commercial apparatus such as the Origen.RTM. 1.5 analyzer by IGEN Inc. permit detection and quantitation of extremely small quantities of analytes in a variety of assays, there is a continuing effort to lower the detection limits and increase the sensitivity of assays performed and also to increase the speed at which assays are performed. Further, it would be advantageous to be able to perform homogeneous immunoassays, or heterogeneous immunoassays requiring fewer separation steps and, thus, less time, upon analytes which heretofore were not amenable to such assays due to the presence of serum matrix components and unbound conjugates which interfere with the desired analyte signal.